Project Summary At the tip of each stereocilium in the hair bundle of vertebrate hair cells is a mechanotransduction complex that mediates the conversion of sound and head movements into the senses of hearing and balance, respectively. TMC1, a ten?pass transmembrane (TM) protein with homology to TMEM16 and OSCA ion channels, has recently been identified as a pore?forming, ion channel subunit of the complex. Protocadherin 15 (PCDH15) and cadherin 23 (CDH23) bind to form a tetrameric, double?stranded protein filament, the `tip link' that physically connects the mechanotransduction complex to the side of a taller, neighboring stereocilia. Deflection of the hair bundle increases tension along the tip link, pulling on the complex to open TMC1. It has recently been shown through co?immunoprecipitation (co?IP) experiments that PCDH15 and TMC1 physically interact, yet it not known specifically how. This project aims to identify individual residues within TMC1 and PCDH 15 that facilitate binding and that enable tension to gate the TMC1 channel. Biolayer interferometry and co?IP analysis of chimeric TMC1 and PCDH15 proteins will reveal which general regions are involved in binding. Site?directed mutagenesis will narrow the binding interface to specific residues. If necessary, cross?linking mass spectrometry will reveal residue?to?residue contacts in intracellular and extracellular loops. If binding occurs within the membrane, a classic tryptophan scanning protocol will be employed on the single TM helix of PCDH 15 to find residues essential for interaction with TMC1. Finally, non?binding TMC1 mutants and controls will be expressed in a TMC1/TMC2 double knockout mouse model to determine if mutants localize properly to the tips of hair bundle stereocilia but fail to mediate mechanotransduction.